Modified nuclease gene CAS12ICS and application thereof

Abstract

The invention discloses a modified nuclease gene CAS12ICS and an application of the modified nuclease gene CAS12ICS. Specifically, the invention discloses a DNA molecule with a nucleotide sequence of SEQ ID No.1, a gene editing vector prepared by using the DNA molecule, and an application of the DNA molecule in improving the mutation efficiency of nuclease MAD7-mediated corn gene editing. According to the invention, the codon-optimized CAS12ICS gene is obtained through screening, a recombinant vector CPB-CAS12ICS containing the gene is constructed based on the codon-optimized CAS12ICS gene, and experiments show that the CAS12ICS gene can obviously improve the mutation efficiency of nuclease MAD7, and the recombinant vector CPB-CAS12ICS constructed by using the gene has good mutation efficiency in corn, and the mutation efficiency is as high as 50%. The method lays a foundation for wide application of nuclease MAD7 in corn, enriches corn genome editing tools, and has a wide application prospect.

Description

Technical Field

[0001] The present invention belongs to the field of gene editing engineering, and specifically relates to a modified nuclease gene CAS12ICS and an application thereof. Background Art

[0002] Genome editing is a technology that uses programmable sequence-specific nucleases to perform targeted and precise modifications on the genome, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats / CRISPR-associated nuclease (CRISPR / Cas). The CRISPR-Cas system has become the most widely used choice in agriculture due to its cost-effectiveness, simplicity, and efficiency. The CRISPR / Cas system is divided into Class I (type I, type III) and Class II (type II, type V, type VI). CRISPR-Cas12a, also known as Cpf1, is a Class II type V CRISPR-Cas system. CRISPR-Cas12a is guided by a single crRNA, catalyzing its own precursor to generate mature crRNA. When the crRNA-Cas12a complex is formed, it recognizes the specific protospacer adjacent motif (PAM) site in the non-target DNA chain. The Ruvc domain cuts the DNA target chain at 8nt from the PAM site, generating a double-strand break (DSB) with a 4-5nt sticky end, activating the DNA damage repair mechanism in the cell, and then generating mutations such as deletions and knock-ins. At present, the CRISPR / Cas12a system is being increasingly used in the targeted editing of different plant genomes such as Arabidopsis, tobacco, rice, wheat, and corn, and has obtained a high induced mutation rate and a genome editing plant that can be stably inherited. MAD7 belongs to the nuclease of the Class II type VA CRISPR family (Cas12a class), which is an engineered variant of the Cas12a nuclease and has 31% homology with the amino acids of the typical AsCpf1. MAD7 has been used for genome editing in various cell lines and species, but has been less widely used in plants.

[0003] Corn (Zea mays L.) is an important food and feed crop, as well as a raw material for the modern food and chemical industries, and plays an important role in my country's agricultural production and economic development. Using gene editing technology to create excellent new germplasm has important research significance. Summary of the invention

[0004] The technical problem to be solved by the present invention is how to improve the mutation efficiency of gene editing mediated by nuclease MAD7, or how to perform efficient gene editing on corn genes. The technical problem to be solved is not limited to the described technical subject matter, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

[0005] In order to solve the above technical problems, the present invention first provides a DNA molecule, which may be named CAS12ICS, and the nucleotide sequence of the DNA molecule may be shown as SEQ ID No.1.

[0006] The DNA molecule may be a codon-optimized gene of the nuclease MAD7, which is a codon-optimized gene encoding the nuclease MAD7 according to maize preference.

[0007] The present invention also provides a biomaterial, which may be any of the following:

[0008] A1) an expression cassette containing the DNA molecule;

[0009] A2) a recombinant vector containing the DNA molecule, or a recombinant vector containing the expression cassette described in A1);

[0010] A3) a recombinant microorganism containing the DNA molecule, or a recombinant microorganism containing the expression cassette described in A1), or a recombinant microorganism containing the recombinant vector described in A2);

[0011] A4) a recombinant host cell containing the DNA molecule, or a recombinant host cell containing the expression cassette of A1), or a recombinant host cell containing the recombinant vector of A2).

[0012] Furthermore, the biological materials can all express the DNA molecules.

[0013] The present invention also provides a recombinant vector, which comprises the DNA molecule, promoter Ubi, promoter U6-2, promoter E35S, a nuclear localization signal, a NOS terminator and a screening marker.

[0014] Furthermore, the nucleotide sequence of the recombinant vector may be as shown in SEQ ID No.2.

[0015] The recombinant vector can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the DNA molecule of the present invention (codon-optimized nuclease gene, SEQ ID No. 1) and can express nuclease MAD7 so that it can perform the cutting function of nuclease MAD7.

[0016] The present invention also provides a gene editing system, which may be the following B1) or B2):

[0017] B1) a gene editing vector, which comprises the DNA molecule and a DNA encoding an sgRNA targeting a target gene;

[0018] B2) A gene editing composition, comprising the following b1) and b2):

[0019] b1) the DNA molecule or any recombinant vector described herein;

[0020] b2) sgRNA targeting the target gene, DNA encoding the sgRNA targeting the target gene, or a recombinant vector expressing the sgRNA.

[0021] The target gene may be a maize gene.

[0022] The recombinant vector expressing the sgRNA may be a recombinant vector containing a DNA encoding the sgRNA.

[0023] The sgRNA can guide the nuclease MAD7 (expressed by the DNA molecule shown in SEQ ID No. 1) to perform gene editing on the target gene.

[0024] The sgRNA may be a sgRNA targeting a corn gene coding region (CDS).

[0025] The structure of sgRNA is well known to those skilled in the art. sgRNA includes a recognition region and a framework region. The framework region is responsible for binding to the Cas protein (nuclease MAD7). The recognition region is responsible for binding to the target site of the target gene (corn gene) (the target site is adjacent to the protospacer adjacent motif (PAM)), guiding the Cas protein (nuclease MAD7) to the target site, and mediating the cutting at a specific site in the MAD7 target site. The design of sgRNA is also well known to those skilled in the art, for example, the online design tool (crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR) can be used for corresponding design.

[0026] Specifically, in the gene editing system of the present invention, sgRNA targets the maize ZmGDIα gene. The sgRNAs targeting the maize ZmGDIα gene may be sgRNA1 and sgRNA2, the target sequence of sgRNA1 may be SEQ ID No.3, and the target sequence of sgRNA2 may be SEQ ID No.4.

[0027] The method for constructing the gene editing system is well known to those skilled in the art. For example, the encoding DNA of the sgRNA targeting the target gene and the DNA molecule of the present invention (SEQ ID No. 1) can be connected to the same vector and started with a dual promoter, or the encoding DNA of the sgRNA targeting the target gene and the DNA molecule of the present invention (SEQ ID No. 1) can be connected to different vectors to construct a gene editing system targeting the target gene.

[0028] In one or more embodiments of the present invention, the gene editing system is a gene editing vector CPB-CAS12ICS-sgRNA (a knockout vector for ZmGDIα), wherein the gene editing vector CPB-CAS12ICS-sgRNA is obtained by cloning a DNA molecule encoding sgRNA1 (SEQ ID No. 3) and a DNA molecule encoding sgRNA2 (SEQ ID No. 4) into a recombinant vector shown in SEQ ID No. 2 (containing a DNA molecule shown in SEQ ID No. 1 of the present invention) (the structure of CPB-CAS12ICS-sgRNA can be as shown in Figure 1 (shown in A).

[0029] Furthermore, the gene editing vector CPB-CAS12ICS-sgRNA is obtained by cloning a DNA molecule having a nucleotide sequence as shown in SEQ ID No. 5 into the recombinant vector CPB-CAS12ICS shown in SEQ ID No. 2, while keeping other nucleotide sequences of the recombinant vector CPB-CAS12ICS unchanged.

[0030] The gene editing vector CPB-CAS12ICS-sgRNA contains two editing target sites of the ZmGDIα gene and the nuclease CAS12ICS gene (SEQ ID No. 1) designed by the present invention. After being introduced into the recipient cells, the two guide RNAs transcribed therefrom can target the ZmGDIα gene through complementary base pairing, and the nuclease MAD7 expressed therefrom causes double-strand breaks of the DNA upstream and downstream of the ZmGDIα gene target site, and connects the sequences upstream and downstream of the break through the organism's own DNA damage repair response mechanism, thereby achieving knockout of the ZmGDIα gene.

[0031] In order to achieve the purpose of site-directed gene mutagenesis, a repair template plasmid (donor DNA molecule) can also be provided. The donor DNA molecule can contain the target gene mutation site and homologous arms located upstream and downstream of the mutation site, which can introduce site-directed mutations during the repair process to achieve site-directed mutations of the target gene.

[0032] It is known to those skilled in the art that the recombinant vector containing the DNA molecule of the present invention, sgRNA, sgRNA expression vector (a vector containing and expressing a DNA molecule encoding sgRNA) and / or donor DNA molecule can be transferred into plant cells by various methods known in the art, for example, it can be transferred into plant recipient cells with a vector as a medium, including but not limited to Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector system and binary vector system) mediated method (Agrobacterium mediated method) and plant virus vector mediated transformation method. It can also be transferred into plant recipient cells by physical or chemical methods, including but not limited to gene gun method (also known as microprojectile bombardment method or biological missile method), chemical stimulation method, electric shock method, liposome mediated method, microinjection method, laser microbeam method, pollen tube channel method, ultrasonic method, air gun method and vortex method. In one or more embodiments of the present invention, the method of introduction is Agrobacterium mediated method.

[0033] In the present invention, sgRNA can be directly introduced into the recipient cell. When an expression vector is used to deliver sgRNA and the DNA molecule of the present invention (SEQ ID No. 1), sgRNA and the DNA molecule of the present invention (SEQ ID No. 1) can be linked to different expression vectors for expression, or can be linked to the same expression vector for expression.

[0034] The present invention also provides the use of the DNA molecule, the biological material, the recombinant vector or the gene editing system in any of the following:

[0035] C1) Application in improving the mutation efficiency of gene editing mediated by nuclease MAD7;

[0036] C2) Application in gene editing mediated by nuclease MAD7;

[0037] C3) Application in the preparation of gene editing products induced by nuclease MAD7.

[0038] The present invention also provides the use of the DNA molecule, the biological material or the recombinant vector in the preparation of a gene editing system mediated by nuclease MAD7 or a gene editing product mediated by nuclease MAD7.

[0039] The method for preparing a gene editing system mediated by nuclease MAD7 using the DNA molecule of the present invention may include the following steps:

[0040] (1) constructing a recombinant vector containing SEQ ID No.1;

[0041] (2) Design sgRNA targeting maize genes;

[0042] (3) Cloning the DNA encoding the sgRNA into the recombinant vector described in step (1) to obtain a gene editing vector, namely, a gene editing system mediated by nuclease MAD7.

[0043] In the above application, the gene editing may be corn gene editing.

[0044] The gene editing product may be gene-edited corn or a new variety of corn.

[0045] The present invention also provides a method for improving the mutation efficiency of corn gene editing mediated by nuclease MAD7 or a gene editing method, the method comprising the step of editing the corn gene using the DNA molecule or any recombinant vector described herein.

[0046] Furthermore, the method may include any of the following:

[0047] D1) constructing the DNA molecule and the DNA molecule encoding the sgRNA into a vector to obtain a gene editing vector, and introducing the gene editing vector into corn to perform gene editing on the corn gene;

[0048] D2) constructing the DNA molecule and the DNA molecule encoding sgRNA into different vectors respectively to obtain a recombinant vector expressing nuclease MAD7 and a recombinant vector expressing sgRNA, and introducing the recombinant vector expressing nuclease MAD7 and the recombinant vector expressing sgRNA into corn to perform gene editing on corn genes;

[0049] The sgRNA can target a maize gene (such as the ZmGDIα gene).

[0050] Furthermore, the method for improving the mutation efficiency of corn gene editing mediated by nuclease MAD7 or the gene editing method may include the following steps:

[0051] (1) constructing a recombinant vector containing SEQ ID No.1;

[0052] (2) Design sgRNA targeting maize genes;

[0053] (3) cloning the DNA encoding the sgRNA into the recombinant vector described in step (1) to obtain a gene editing vector;

[0054] (4) introducing the gene editing vector into corn;

[0055] (5) Obtain corn with gene mutation or gene knockout through screening and identification.

[0056] Furthermore, the introduction method includes but is not limited to: Agrobacterium-mediated method, plant virus vector-mediated transformation method, gene gun method (also known as microparticle bombardment method or biological missile method), chemical stimulation method, electric shock method, liposome-mediated method, microinjection method, laser microbeam method, pollen tube channel method, ultrasonic method, air gun method and vortex method.

[0057] Furthermore, the introduction can be introduced through Agrobacterium-mediated method, and the introduction may include the following steps: introducing the gene editing vector obtained in step (3) into Agrobacterium to obtain recombinant Agrobacterium; infecting corn callus or explants with the recombinant Agrobacterium; and inducing and culturing the obtained positive callus or explants to obtain regenerated plants after identification.

[0058] The explants include, but are not limited to, seeds, roots, leaves, petioles, cotyledons, cotyledon petioles, hypocotyls, stem segments, stem apical meristems, epidermal parenchyma cells, tubers, stolon segments, embryonic suspension cells, and protoplasts.

[0059] The screening and identification methods are known to those skilled in the art. For example, gene-edited plants (including offspring materials of gene-edited plants) can be identified by PCR detection, Sanger sequencing, high-throughput sequencing, Western Blot, Southern blot hybridization, and the like.

[0060] Further, the corn gene can be a nucleic acid molecule encoding a corn protein of interest (any protein of interest). For example, the corn gene can be a gene that is isolated, purified, cloned and transformed into an organism that can bring about an expected phenotypic trait (such as insect resistance or herbicide tolerance). In one or more embodiments of the present invention, the corn gene is a ZmGDIα gene.

[0061] Furthermore, the sgRNA targets the maize gene coding region (CDS), and can be conventionally designed by a person skilled in the art according to the design rules of sgRNA. For example, an online design tool (crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR) can be used for corresponding design.

[0062] Furthermore, the recombinant vector in step (1) may also include a promoter (such as promoters Ubi, U6, U6-2, E35S, etc.), a nuclear localization signal (NLS), a terminator (such as NOS terminator) and a selection marker.

[0063] The selection marker can be selected from antibiotic resistance genes (such as ampicillin resistance gene (Amp r ), tetracycline resistance gene (Tet r ), chloramphenicol resistance gene (Cmp r ), kanamycin resistance gene (Kanamycin r Neomycin resistance gene (Neo r ), hygromycin resistance gene (hyg), streptomycin resistance gene (str), bleomycin resistance gene (ble), etc.) and herbicide resistance genes (such as bar gene, glyphosate resistance marker gene epsps, chlorsulfuron resistance marker gene als, etc.).

[0064] In one or more embodiments of the present invention, the recombinant vector containing SEQ ID No.1 is the recombinant vector shown in SEQ ID No.2; the corn gene is the ZmGDIα gene; and the sgRNA includes sgRNA1 (target sequence is SEQ ID No.3) and sgRNA2 (target sequence is SEQ ID No.4).

[0065] The gene editing described herein may include gene knockout, gene knock-in, gene mutation, gene fragment replacement or gene modification.

[0066] The gene editing system described in this article can be a MAD7-mediated gene editing system (CRISPR / MAD7 gene editing system).

[0067] The present invention designs different codon optimization schemes for corn, and unexpectedly obtains a codon-optimized nuclease MAD7 gene (CAS12ICS gene, SEQ ID No. 1) capable of greatly improving the editing efficiency of nuclease MAD7 after screening, and based on this, constructs a recombinant vector CPB-CAS12ICS containing the CAS12ICS gene, and experimental results show that the CAS12ICS gene of the present invention can significantly improve the mutation efficiency of nuclease MAD7, and the recombinant vector CPB-CAS12ICS constructed using the CAS12ICS gene has good mutation efficiency in corn, with a mutation efficiency of up to 50%. The present invention lays a foundation for the wide application of nuclease MAD7 in corn, enriches corn genome editing tools, and has broad application prospects.

[0068] Definition of terms

[0069] In the present invention, unless otherwise specified, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Meanwhile, in order to better understand the present invention, the definitions and explanations of the relevant terms are provided below.

[0070] The term "expression cassette" generally refers to a nucleic acid construct comprising nucleic acid elements sufficient to express a target gene. A typical expression cassette comprises a promoter, an MCS (multiple cloning site) and a terminator. The expression cassette may also include a target gene, a marker gene (such as a TK gene, a DHFR gene, a CAT gene and a NEO gene), a ribosome recognition and binding site (SD), a transcription factor binding site (TFBS), an enhancer, a silencer, a repressor, an intron, a poly (A) signal sequence and / or an mRNA splicing signal sequence, etc. The elements in the expression cassette may be directly connected or indirectly connected via a linker.

[0071] The term "vector" generally refers to a vector that can carry foreign DNA or target gene into host cells for amplification and / or expression. The vector can be a cloning vector or an expression vector. The vector can be introduced into host cells by transformation, transduction or transfection, so that the genetic material elements it carries are amplified and / or expressed in the host cells. Those skilled in the art can select a suitable vector according to the purpose of genetic engineering and the properties of the recipient cells. The vector includes, but is not limited to, plasmid, phage (e.g., lambda phage or M13 phage), cosmid, phagemid, shuttle vector (e.g., yeast expression vector), Ti plasmid, artificial chromosome (e.g., yeast artificial chromosome (YAC), bacterial artificial chromosome (BAC), P1 artificial chromosome (PAC), or Ti plasmid artificial chromosome (TAC)), viral vector (e.g., baculovirus vector, retrovirus (including lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, papillomavirus (e.g., SV40), herpesvirus (e.g., herpes simplex virus)). A vector may contain a variety of elements for controlling expression, including, but not limited to, promoter sequence, transcription initiation sequence, enhancer sequence, selection element, and reporter gene. In addition, the vector may also contain a replication initiation site.

[0072] The term "microorganism" generally includes bacteria, viruses, fungi, actinomycetes, rickettsia, mycoplasma, chlamydia, spirochetes, algae, etc. For example, the bacteria may be from Corynebacterium sp. (e.g., Corynebacterium glutamicum, Corynebacterium pekinensis, Corynebacterium dentata, etc.), Brevibacterium sp. (e.g., Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium ammoniaphagoides, etc.), Escherichia sp. (e.g., Escherichia coli), Erwinia sp., Agrobacterium sp. (e.g., Agrobacterium tumefaciens), Flavobacterium sp., Alcaligenes sp., Pseudomonas sp., and Bacillus sp. (e.g., Bacillus sp.), etc. The virus may include rotavirus, baculovirus, retrovirus (such as lentivirus), adenovirus, adeno-associated virus, poxvirus, papillomavirus, influenza virus, papillomavirus (such as SV40) and herpes virus (such as herpes simplex virus), etc. The fungus may be from Saccharomyces sp. (such as saccharomyces cerevisiae, Candida, methanol yeast, Pichia pastoris), Fusarium sp., Rhizoctonia sp., Verticillium sp., Penicillium sp., Aspergillus sp. and Cephalosporium sp., etc. The actinomycete may be from Streptomyces sp. (such as Streptomyces). The algae may be from Cyanophyta (such as Cyanobacteria), Fucus sp., Achnanthes sp., Amphiprora sp., Amphora sp., Ankistrodesmus sp., Asteromonas sp., Boekelovia sp., and the like.

[0073] The term "host cell" is also referred to as a recipient cell, and generally refers to any type of cell that can be used to introduce a vector, such as a plant cell and an animal cell. The host cell can be understood to refer not only to a specific recipient cell, but also to the progeny of such a cell, and due to natural, accidental or intentional mutations and / or changes, the progeny may not necessarily be completely identical to the original parent cell, but is still included in the scope of the host cell. Suitable host cells are known in the art, wherein: the plant cell may be Arabidopsis thaliana, tobacco (Nicotiana tabacum), corn (Zea mays), rice (Oryza sativa), wheat (Triticum aestivum) and the like plant cells but are not limited thereto; the animal cell may be a mammalian cell (e.g., Chinese hamster ovary cell (CHO cell), Chinese hamster ovary cell substrain (CHO-K1 cell), African green monkey kidney cell (Vero cell), SV40-transformed African green monkey kidney cell (COS cell), baby hamster kidney cell (BHK cell), mouse breast cancer cell (C127 cell), human embryonic kidney cell (HEK293 cell), human HeLa cell, fibroblast, bone marrow cell line, T cell or NK cell, etc.), avian cell (e.g., chicken or duck cell), amphibian cell (e.g., African clawed frog (Xenopus laevis) cell or giant salamander (Andrias davidianus) cells), fish cells (such as grass carp, carp, rainbow trout or catfish cells), insect cells (such as Sf21 cells, Sf-9 cells or Hi-5), etc., but are not limited thereto.

[0074] The term "recombinant vector" generally refers to a recombinant DNA molecule constructed by connecting an exogenous target gene to a vector in vitro. It can be constructed in any suitable manner, as long as the constructed recombinant vector can carry the exogenous target gene into the recipient cell and provide the exogenous target gene with the ability to replicate, integrate, amplify and / or express in the recipient cell.

[0075] The term "recombinant microorganism" generally refers to a recombinant microorganism with changed functions obtained by manipulating and modifying the genes of a target microorganism, such as introducing an exogenous target gene or a recombinant vector into the target microorganism, or directly editing the endogenous genes of the target microorganism.

[0076] The term "recombinant host cell" generally refers to a recombinant host cell whose function has been changed by manipulating and modifying the genes of the host cell, such as introducing an exogenous target gene or a recombinant vector into the host cell, or directly editing the endogenous genes of the host cell.

[0077] The term "introduction" generally refers to the transfer of foreign genes into recipient cells such as eukaryotic recipient cells or prokaryotic recipient cells. The method of introduction is not particularly limited, and any known transformation method can be used as long as the target gene (such as the DNA molecule of the present invention) can be transferred into the recipient cell. The introduced DNA molecule can be a single copy or multiple copies. The introduction can be the integration of the foreign gene into the host chromosome, or it can be expressed extrachromosomally by a plasmid. The method of introduction can include any of the following: (1) introducing the target gene or a recombinant vector containing the target gene into the host bacteria by chemical transformation (such as Ca ion-induced transformation, polyethylene glycol-mediated transformation, or metal cation-mediated transformation, etc.) or physical transformation (such as electroporation transformation). (2) transducing the target gene into the host bacteria by phage transduction. (3) The target gene is transferred into plant receptor cells by physical or chemical methods, such as gene gun method (also known as microprojectile bombardment method or biological missile method), chemical stimulation method, electric shock method, liposome-mediated method, microinjection method, laser microbeam method, pollen tube channel method, ultrasound method, air gun method and vortex method, etc. (4) The target gene is transferred into plant receptor cells by vector, such as Agrobacterium Ti plasmid vector (including Ti plasmid-derived vectors such as co-integration vector system and binary vector system) mediated method (Agrobacterium-mediated method), plant virus vector-mediated transformation method, etc. (5) The target gene is introduced into in vitro animal cells (transfection) by calcium phosphate coprecipitation method, cationic polymer method (such as DEAE-dextran transfection method), cationic liposome method, electroporation method (i.e. electrofection method), microinjection, gene gun method or virus-mediated method (such as retrovirus infection method, adenovirus infection method, lentivirus infection method), etc. (6) Introducing the target gene into in vivo animal cells through microinjection, retroviral vector method, somatic cell nuclear transfer method, sperm vector method or embryonic stem cell method, and further preparing transgenic animals.

[0078] The term "codon optimization" generally refers to the process of optimizing the target gene by mainly using preferred codons and avoiding rare codons with low utilization rates without changing the amino acids of the protein. Different species may use different codons to translate the same amino acid, and codon preferences vary from species to species. Codon optimization usually redesigns genes by avoiding rare codons, using preferred codons, simplifying the secondary structure of mRNA, optimizing repetitive sequences, eliminating restriction enzyme sites, and adjusting GC content to improve protein performance.

[0079] The term "target gene (or target gene)" generally refers to a nucleic acid molecule encoding a target protein (any protein of interest). In the gene cloning process, the target gene can be the gene that is to be isolated, purified, cloned and transformed into an organism that can bring about the expected phenotypic trait (such as insect resistance or herbicide tolerance).

[0080] The term "gene editing" generally refers to the ability to change specific gene sequences in any cell, including somatic cells, and can cause gene base deletions, duplications, insertions, frameshift mutations, and replacement and knockout of target genes, achieving replacement, deletion, splicing, and single-base changes in genomic sequences, that is, the technology of arbitrarily "editing" the genome or the sequence of a specific gene.

[0081] The term "DNA encoding sgRNA" generally refers to a DNA molecule that can be transcribed to generate the sgRNA. It is well known to those skilled in the art that, if the sequence of an sgRNA is given, the U in the sgRNA sequence can be replaced by T, and the other nucleotides remain unchanged to obtain a DNA sequence encoding the sgRNA.

[0082] The term "sgRNA (single-guide RNA, single-guide RNA)" generally refers to the structure of a single RNA formed by artificially modifying a crRNA / tracrRNA complex (gRNA) with a dual RNA structure, and directly connecting crRNA and tracrRNA (or through a connector). In some CRISPR-Cas systems, the Cas protein is guided by only a single crRNA molecule, rather than a crRNA / tracrRNA complex, for example, Cas12a, Cas12i3, Cas13a, Cas13b, and the Cas protein (MAD7) of the present invention do not require an effective tracrRNA. In such systems, sgRNA is equivalent to crRNA, rather than a crRNA / tracrRNA complex. The nuclease MAD7 of the present invention and the Cas12a protein both belong to Class II V-type A subtype CRISPR-Cas systems. sgRNA is a component of the CRISPR-Cas system, responsible for guiding the Cas protein to recognize and cut the target nucleic acid molecule. In actual gene editing applications, sgRNA can be directly synthesized or obtained by plasmid expression or in vitro transcription.

[0083] The term "target sequence" herein refers to the target sequence of sgRNA, which refers to a sequence located in the gene to be edited (usually the coding region), which is complementary to the sgRNA, located near the desired location for cutting, and is the target site recognized by the sgRNA. The target sequence can determine the location and specificity of gene editing.

[0084] The term "recognition region" may also be referred to as a guide sequence, which is generally a targeting sequence contained in a guide RNA (sgRNA or gRNA). The guide sequence is generally sufficiently complementary to the target sequence so that it can hybridize with the target sequence and guide the CRISPR / Cas complex to specifically bind to the target sequence. Complete complementarity between the guide sequence and the target sequence is preferred, but certain mismatches (such as mismatches of 1-6 nucleotides) are also allowed, as long as they can still lead to knockout of the target gene. The degree of complementarity between the guide sequence and its corresponding target sequence is at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or at least 99%. The method for determining the complementarity of two nucleic acid sequences is within the capabilities of those of ordinary skill in the art.

[0085] The term "scaffold" is generally a sequence in a guide RNA (sgRNA or gRNA) that is recognized and bound by a Cas protein, and may also be referred to as a backbone sequence of an sgRNA. Scaffolds may be routinely selected by a person skilled in the art.

[0086] The term "explant" generally refers to a part of a plant body used as an in vitro culture material in plant tissue culture, which can be regenerated into a whole plant under appropriate conditions after appropriate treatment. In actual operation, those skilled in the art select appropriate explants for transformation according to different plants.

[0087] The term "callus" usually refers to the newly formed tissue on the wound surface after the original plant body is partially stimulated by trauma. It is composed of living thin-walled cells and can originate from living cells of various tissues in any organ of the plant body. In plant tissue culture, it can refer to a group of disorderly growing thin-walled cells with vigorous division ability formed by explants. Cultivating callus on an appropriate culture medium can induce it to form a whole plant. BRIEF DESCRIPTION OF THE DRAWINGS

[0088] Figure 1 Knockout vector and mutation identification for the ZmGDIα gene. Figure 1 A and B are schematic diagrams of the structure of the knockout vector CPB-CAS12ICS-sgRNA in Example 3; Figure 1 Middle C is the mutation identification result. DETAILED DESCRIPTION

[0089] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0090] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0091] The maize inbred line KN5585 in the following examples is derived from Weimi Biotechnology (Jiangsu) Co., Ltd.

[0092] The composition of the N6 liquid medium in the following example is: potassium nitrate 2800 mg / L, ammonium sulfate 463 mg / L, potassium dihydrogen phosphate 400 mg / L, magnesium sulfate 185 mg / L, calcium chloride 165 mg / L, potassium iodide 0.8 mg / L, boric acid 1.6 mg / L, manganese sulfate monohydrate 4.4 mg / L, zinc sulfate heptahydrate 1.5 mg / L, disodium ethylenediaminetetraacetic acid 37.25 mg / L, ferrous sulfate heptahydrate 27.85 mg / L, glycine 2 mg / L, vitamin B1 1 mg / L, vitamin B6 0.5 mg / L, nicotinic acid 0.5 mg / L, sucrose 20000 mg / L. Weigh 24.1 g in 1000 mL of distilled water or deionized water, heat and stir to dissolve, adjust the pH to 5.8±0.2 (25° C.), and sterilize at 115° C. for 20 minutes.

[0093] Example 1. Optimal design of nuclease MAD7

[0094] According to the degeneracy of codons and the preference of amino acid codons, and with reference to the nuclease MAD7 protein sequence published in Genebank (GenBank accession number: WP_055225123.1), the inventors designed an optimized nuclease MAD7 coding gene containing maize preferred codons. After screening, they unexpectedly obtained a codon sequence of MAD7 that can greatly improve the nuclease editing efficiency (mutation efficiency). The nuclease MAD7 gene after codon optimization for expression in maize was named CAS12ICS gene, and the nucleotide sequence of the CAS12ICS gene is shown in SEQ ID No. 1.

[0095] Example 2: Construction of recombinant vector

[0096] A recombinant vector was constructed based on the nuclease gene (CAS12ICS gene) designed in Example 1, and the recombinant vector includes the following elements: promoter, CAS12ICS gene, nuclear localization signal (NLS), terminator and screening marker. Using CPB as the backbone vector, a recombinant vector based on CRISPR / CAS12ICS was constructed and named CPB-CAS12ICS. The constructed recombinant vector CPB-CAS12ICS includes the following elements from N-terminus to C-terminus: a gene sequence encoding MAD7 protein driven by the Ubi promoter. The bar screening gene driven by the CaMV 35S promoter; the KAN antibiotic resistance gene; the replication element; in the transgenic positive plants, the MAD7 protein expressed by Ubi::MAD7::Nos on the MAD7 vector has directional DNA cleavage activity after binding to the sgRNA transcribed by U6::scaffold::sgRNA::HDV; the bar protein expressed by CaMV 35S::bar::CaMV poly(A)signal has herbicide resistance and can be used for the screening of positive plants. The nucleotide sequence of the recombinant vector CPB-CAS12ICS is shown in SEQ ID No. 2. Wherein:

[0097] The 115th to 367th positions of SEQ ID No.2 are NOS terminator; the 393th to 458th positions of SEQ ID No.2 are nuclear localization signal 1 (NLS1); the 459th to 4244th positions of SEQ ID No.2 are CAS12ICS gene (reverse complementary to the sequence shown in SEQ ID No.1); the 4245th to 4295th positions of SEQ ID No.2 are nuclear localization signal 2 (NLS2); the 4296th to 4361th positions of SEQ ID No.2 are 3×flag; the 4367th to 6338th positions of SEQ ID No.2 are promoter Ubi; the 13069th to 13863th positions of SEQ ID No.2 are screening marker KAR (kanamycin resistance gene); the 14390th to 14564th positions of SEQ ID No.2 are CaMV poly (A) signal; the 14571th to 15122th positions of SEQ ID No.2 are screening marker Bar; Positions 15167-15843 of No. 2 are CaMV 35S promoter (enhanced).

[0098] Example 3: Verification of mutation efficiency of recombinant vector CPB-CAS12ICS

[0099] 1. Design sgRNA and promoter

[0100] In order to verify the mutation efficiency of the recombinant vector CPB-CAS12ICS, two sgRNAs were designed based on the ZmGDIα (Zm00001d010255) gene in the reference genome sequence B73_RefGen_v4 to knock out the ZmGDIα gene.

[0101] Target sequence of sgRNA1: 5'-AGCCCCGTGCCCAGAACGATC-3' (SEQ ID No. 3),

[0102] Target sequence of sgRNA2: 5′-GGGAGGTGGAATCTCCTCCGT-3′ (SEQ ID No. 4).

[0103] The sgRNA sequence was verified by Sanger sequencing in the maize inbred line KN5585 recipient.

[0104] The sgRNA was driven by the endogenous U6-2 promoter of maize.

[0105] 2. Construction of knockout vector

[0106] First, the recombinant vector CPB-CAS12ICS was digested with HindIII restriction endonuclease. The reaction was carried out in a constant temperature metal bath at 37°C to ensure the maximization of enzyme activity. The entire reaction process lasted for 3 hours. After the reaction was completed, 1.2% agarose gel electrophoresis was performed to separate and identify different DNA fragments. After the electrophoresis, with the precise positioning of the UV irradiator, the gel portion containing only the target DNA fragment was carefully cut off to ensure the purity and integrity of the CPB-Cas12ICS linear vector in the subsequent recovery step. Finally, the cut gel portion was finely recovered to efficiently extract the CPB-Cas12ICS linear vector.

[0107] Obtaining the connection fragments:

[0108] First, the homology arm amplification primers were designed, and U6-1-F (5' end containing the vector backbone sequence) and U6-R were used to amplify U6-2. Since the sgRNA is only 20bp and its 5' end is connected to U6-2, and its 3' end is connected to the scaffold sequence, that is, DMSG-1F contains the 3' end of U6-2, 20bp sgRNA and the 5' end of the scaffold, DMSG-1F and sgRNA-R (3' end containing the vector backbone sequence) were used to amplify the target and scaffold sequences. Then, Phanta Max Super-Fidelity DNA Polymerase and the designed primers were used to amplify the U6-2 sequence, the target, and the scaffold sequence, and then the gel was cut and recovered. Since the U6-2 sequence and the target sequence have homology arms, the two fragments were overlapped PCR after recovery, so that the U6-2 sequence, the target sequence, and the scaffold sequence overlap into one fragment. Finally, the CPB-Cas12ICS linear vector and the overlapping fragments were connected with the NEB Builder homologous recombination enzyme.

[0109] The nucleotide sequence of the overlapping fragment is shown in SEQ ID No. 5. Wherein: SEQ ID No. 5 No. 1-397 is U6-2. SEQ ID No. 5 No. 442-476 is the scaffold sequence. SEQ ID No. 5 No. 477-497 is the target sequence of sgRNA1. SEQ ID No. 573-969 is U6-2. SEQ ID No. 5 No. 1014-1048 is the scaffold sequence. SEQ ID No. 5 No. 1049-1069 is the target sequence of sgRNA2.

[0110] The knockout vector constructed above is named CPB-CAS12ICS-sgRNA, and its structure diagram is shown in Figure 1 As shown in A.

[0111] The knockout vector CPB-CAS12ICS-sgRNA is obtained by cloning a DNA molecule having a nucleotide sequence as shown in SEQ ID No.5 into a recombinant vector CPB-CAS12ICS (SEQ ID No.2), while keeping other nucleotide sequences of the recombinant vector CPB-CAS12ICS unchanged.

[0112] The knockout vector CPB-CAS12ICS-sgRNA contains two editing target sites of the ZmGDIα gene and the nuclease CAS12ICS gene designed by the present invention (SEQ ID No. 1). After being introduced into the recipient cells, the two guide RNAs transcribed therefrom can target the ZmGDIα gene through complementary base pairing, and the nuclease MAD7 expressed therefrom causes DNA double-strand breaks upstream and downstream of the ZmGDIα gene target site (such as Figure 1 The DNA damage repair response mechanism of the organism itself connects the upstream and downstream sequences of the break, thereby achieving the knockout of the ZmGDIα gene ( Figure 1 Middle C).

[0113] 3. Maize genetic transformation

[0114] (1) The constructed knockout vector CPB-CAS12ICS-sgRNA was introduced into BMEHA105 Agrobacterium competent cells (Beijing Bomade Gene Technology Co., Ltd., catalog number: BC303-01) to obtain recombinant Agrobacterium.

[0115] (2) Using N6 liquid medium to culture the recombinant Agrobacterium obtained in step (1), the bacterial solution OD 600nm The recombinant Agrobacterium liquid was centrifuged at 5000r / min for 10min, the cells were collected, and the cells were suspended in the prepared infection buffer (1L infection buffer is prepared by mixing 4g of N6 salt containing N6 vitamin, 2mg 2,4-D, 100mg inositol, 0.7g L-proline, 68.4g sucrose, 36g glucose, 1mL AgNO3 (10mg / mL), 1mL As (100mol / L) and water, pH 5.2) to make the OD 600nm The value is about 0.5, and then it is shaken at 28℃ and 150r / min for 0.5h to obtain the infection solution.

[0116] (3) Soak the well-growing callus tissue (maize embryo) of the maize inbred line KN5585 in the infection buffer for 1 hour, then transfer it to the infection solution prepared in step (2) and soak it for 15 minutes, and then dry it.

[0117] (4) The callus infected in step (3) was placed in a co-culture medium (1 L of co-culture medium was prepared by mixing 4 g of N6 salt containing N6 vitamin, 2 mg of 2,4-D, 30 g of sucrose, 8 g of agar, 1 mL of AgNO3 (10 mg / mL), 1 mL of As (100 mol / L), 3 mL of L-cysteine ​​(100 mg / mL) and water, pH 5.8), cultured at 20°C for 3 days, and then transferred to a recovery medium (1 L of recovery medium was prepared by mixing 4 g of N6 salt, 1 ml of N6 vitamin 1000×, 1.5 mg of 2,4-D, 0.7 g of L-proline, 30 g of sucrose, 5 μM AgNO3, 0.5 g of MES, 100 mg of cefotaxime, 100 mg of vancomycin, 8 g of agar and water, pH 5.8). 5.8), cultured at 28°C for 10 days, then transferred to recovery medium containing 1.5 mg / L glufosinate-ammonium, cultured in the dark at 28°C for 7 days, and positive callus was selected.

[0118] (5) The positive callus obtained in step (4) is transferred to an embryoid induction medium (1L embryoid induction medium is obtained by mixing 4.43g of MS salt containing MS vitamin (containing inositol), 0.25mg 2,4-D, 30g sucrose, 5mg 6-BA, 4g plant gel, 1mL Cefo (250mg / mL) and water, pH 5.8), cultured in the dark for 2 weeks, and then transferred to a differentiation medium (1L differentiation medium is obtained by mixing 4.43g of MS salt containing MS vitamin (containing inositol), 30g sucrose, 4g plant gel, 1mL Cefo (250mg / mL) and water, pH 5.8). After green seedlings grow, the culture medium is transferred to a rooting medium (1L rooting medium is obtained by mixing 2.22g MS, 30g sucrose, 51.55mg MS vitamin, 4g plant gel and water, pH 5.8) After the roots have grown to a certain height, they are exposed to the air for 3 days and then transplanted.

[0119] (6) Take a leaf of about 3 cm from the plant, put it into a tube and grind it thoroughly, add 500 μl of buffer (from the bar gene test strip), and insert it into the bar gene test strip (Beijing Aochuang Jinbiao Biotechnology Co., Ltd., product number: A07-13-413). The plant with a positive band is the T0 generation positive plant.

[0120] 4. Verification of genetically modified ingredients

[0121] DNA was extracted and purified using a plant genomic DNA extraction kit (Tiangen, China). T-DNA was detected by Bar test strips (Agdia, Cat. # STX14200 / 0012, US) and PCR of the SpCas9 gene. SpCas9 PCR amplification used a forward primer of 5'-CAACCGGAAAGTGACCGTGA-3' and a reverse primer of 5'-CACCACCTTCACTGTCTGCA-3'. The PCR program included 94°C for 3 min; 35 cycles of 95°C for 30 s, 58°C for 30 s, and 68°C for 20 s; and a final extension of 68°C for 10 min.

[0122] 5. Mutation Detection

[0123] Primers were designed according to the target position of the knockout vector, and the target gene was amplified by PCR in the corresponding plants. After sequencing, the mutation type was analyzed using DSDecode software. The PCR reaction system and reaction procedure were the same as those for the transgenic component verification. Among them, the sequence-specific PCR primers for the target site of the ZmGDIα (Zm00001d010255) gene were 5'-GCACCGCCTGATCTACTACC-3' and 5'-CAGTCTAGGGAAGCCCTCGC-3'.

[0124] 5. Results and Analysis

[0125] The present invention uses the designed and optimized CAS12ICS gene (SEQ ID No. 1) to construct a gene editing vector (CPB-CAS12ICS, SEQ ID No. 2), and uses the gene editing vector to successfully construct a gene editing vector (CPB-CAS12ICS-sgRNA, Figure 1 A and B in Figure 2). Through Agrobacterium-mediated genetic transformation, a positive transformation event was obtained. Through PCR amplification and Sanger sequencing, it was found that a mutation occurred in the sgRNA region and a frameshift mutation was obtained ( Figure 1 C). The types of mutations include the reduction of 10 bases and 25 bases in the ZmGDIα gene, and the mutation efficiency is as high as 50%. This shows that the gene editing vector CPB-CAS12ICS constructed by the present invention can achieve the gene editing function of efficient mutation in a specific genomic region.

[0126] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. A DNA molecule, characterized in that The nucleotide sequence of the DNA molecule is shown as SEQ ID No.

1.

2. Biomaterial, characterized in that The biological material is any of the following: A1) an expression cassette containing the DNA molecule according to claim 1; A2) a recombinant vector containing the DNA molecule of claim 1, or a recombinant vector containing the expression cassette of A1); A3) a recombinant microorganism containing the DNA molecule of claim 1, or a recombinant microorganism containing the expression cassette of A1), or a recombinant microorganism containing the recombinant vector of A2); A4) a recombinant host cell containing the DNA molecule of claim 1, or a recombinant host cell containing the expression cassette of A1), or a recombinant host cell containing the recombinant vector of A2).

3. A recombinant vector, characterized in that: The recombinant vector comprises the DNA molecule according to claim 1, promoter Ubi, promoter U6-2, promoter E35S, a nuclear localization signal, a NOS terminator and a screening marker.

4. The recombinant vector according to claim 3, characterized in that The nucleotide sequence of the recombinant vector is shown as SEQ ID No.

2.

5. A gene editing system, characterized in that: The gene editing system is the following B1) or B2): B1) a gene editing vector, comprising the DNA molecule of claim 1 and a DNA encoding an sgRNA targeting a target gene; B2) A gene editing composition, comprising the following b1) and b2): b1) the DNA molecule according to claim 1 or the recombinant vector according to any one of claims 2 to 4; b2) sgRNA targeting the target gene, DNA encoding the sgRNA targeting the target gene, or a recombinant vector expressing the sgRNA.

6. Use of the DNA molecule according to claim 1, the biological material according to claim 2, the recombinant vector according to claim 3 or 4, or the gene editing system according to claim 5 in any of the following: C1) Application in improving the mutation efficiency of gene editing mediated by nuclease MAD7; C2) Application in gene editing mediated by nuclease MAD7; C3) Application in the preparation of nuclease MAD7-mediated gene editing products.

7. Use of the DNA molecule according to claim 1, the biological material according to claim 2, or the recombinant vector according to claim 3 or 4 in the preparation of a gene editing system mediated by nuclease MAD7 or a gene editing product mediated by nuclease MAD7.

8. The use according to claim 6 or 7, characterized in that: The gene editing is corn gene editing.

9. A method for improving the mutation efficiency of maize gene editing mediated by nuclease MAD7 or a gene editing method, characterized in that: The method comprises the step of editing a corn gene using the DNA molecule of claim 1 or the recombinant vector of any one of claims 2 to 4.

10. The method according to claim 9, characterized in that The method comprises any of the following: D1) constructing the DNA molecule of claim 1 and the DNA molecule encoding the sgRNA into a vector to obtain a gene editing vector, and introducing the gene editing vector into corn to perform gene editing on the corn gene; D2) constructing the DNA molecule according to claim 1 and the DNA molecule encoding sgRNA into different vectors respectively to obtain a recombinant vector expressing nuclease MAD7 and a recombinant vector expressing sgRNA, and introducing the recombinant vector expressing nuclease MAD7 and the recombinant vector expressing sgRNA into corn to perform gene editing on corn genes; The sgRNA targets a maize gene.

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